Project Summary Factor XIII is found in quite different locations including plasma (A2B2), the cellular (A2) of platelets, megakaryocytes, and monocytes, and (A2) in bone extracellular matrix. Plasma FXIII A2B2 is activated proteolytically by thrombin and low mM Ca2+ whereas FXIII A2 is activated nonproteolytically by a range of Ca2+ concentrations. The resultant transglutaminases (FXIII A* and A) introduce covalent ?-glutamyl-?-lysyl crosslinks into protein networks. The main objective of our research is to decipher the kinetic and conformational differences between the two activated forms of Factor XIII (FXIII) and to characterize their ability to affect fibrin clot architecture and related processes. FXIII has roles in hemostasis, venous thrombosis, cardiac repair, bone remodeling, innate immunity, etc. There are, however, few examples of strategies to therapeutically target FXIII. To further enhance such developments, critical gaps in knowledge need to be addressed. We need to know more about 1) the substrate specificities and structural features of activated FXIII species from different activation pathways and 2) how fibrin clot architecture is controlled by FXIII catalyzed crosslinking reactions involving the fibrin ?C region and ? module. The proposed research will thus address two hypotheses: 1) Factor XIII is hypothesized to have optimized activation pathways, protein structural features, and substrate specificities to effectively target its actions in different physiological environments. Research will focus a) on the kinetic properties of proteolytic FXIII A* and nonproteolytic FXIII A, b) on probing conformational roles of FXIII Activation Peptide interactions, and c) on enhancing calcium binding. 2) The fibrin(ogen) ?C region and ? module are hypothesized to enhance their interactions with FXIII by utilizing distinct reactive glutamine environments and employing binding segments for FXIII. a) With ?C (233-425), we will focus on quantitatively exploring how individual reactive Qs will facilitate the reactivities of remaining Qs. b) With ?-module (142-411), we will rank the reactivities of Q398/Q399 and assess the role that anchoring sites plays in controlling substrate specificity. The biochemical methods required to address these aims include activity assays, fibrin crosslinking gels, mass spectrometry, solution NMR, and analytical ultracentrifugation. Our contributions to this research field will have a positive impact because they will help to address unresolved issues associated with FXIII structure-function, and as a result, lead to new innovative strategies to manage FXIII in particular physiological locations